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Sociobiology 65(4): 645-653 (October, 2018) Special Issue DOI: 10.13102/.v65i4.3464

Sociobiology An international journal on social

Research article -

Two Colors, One : The Case of nigroaenea (: ), an Important of Cotton Fields in Brazil

C Grando1,2, ND Amon2,6, SJ Clough3, N Guo3, W Wei3, P Azevedo1,4, MM López-Uribe2, MI Zucchi5

1 - Institute of , University of Campinas, Campinas, São Paulo, Brazil 2 - Department of Entomology, Center for Pollinator Research, Pennsylvania State University, University Park, USA 3 - Department of Crop , University of Illinois at Urbana-Champaign, Urbana, USA 4 - Centro de Estudos em Insetos Sociais, Universidade Estadual Paulista “Julio de Mesquita Filho”, Rio Claro, São Paulo, Brazil 5 - Agência Paulista de Tecnologia dos Agronegócios, Pólo Regional Centro-Sul, Piracicaba, São Paulo, Brazil 6 - Department of Entomology, University of Wisconsin-Madison, Madison, USA

Article History Abstract Accurate taxonomic delimitation in ecological research is absolutely critical as Edited by studies that seek to evaluate levels of and quantify effects on Eduardo Almeida, USP, Brazil Received 10 May 2018 the environment are rapidly undertaken. Coloration is a widely used morphological Initial acceptance 02 July 2018 character for species through dichotomous keys. However, taxonomic Final acceptance 26 August 2018 identification based upon coloration is often unreliable because character can Publication date 11 October 2018 exhibit high degree of intraspecific variation. An uncertain interpretation of mesosoma color-morphs (yellow or black) occurred when we used this character, in association Keywords Melissodes Cytochrome oxidase I, Bayesian with the bristle bands in the TII, for the identification of eucerine phylogeny, bees, barcoding gap. nigroaenea (Smith), an important pollinator of cotton fields in South America. Thus, we used a DNA barcoding approach to investigate if different color morphs within Corresponding author this species reflected two distinct evolutionary lineages. Our Bayesian phylogenetic Maria I. Zucchi, reconstruction revealed that both yellow and black individuals clustered in a highly Agência Paulista de Tecnologia supported monophyletic group. Additionally, pairwise genetic distances between dos Agronegócios Pólo Regional Centro Sul M. nigroaenea color-morphotypes were lower than 3%. These results indicate that Rodovia SP 127, km 30 both mesosoma color-morphs correspond to intraspecific variability within the same CEP 13400-970, Piracicaba-SP, Brasil. evolutionary unit. Although the mesosoma coloration is not the unique character E-Mail: [email protected] considered for taxonomic differentiation among Melissodes species, our results indicate that this character is not reliable for Melissodes species differentiation. The incorporation of DNA barcoding approaches to taxonomic classification can help resolve some of the problems that originate while relying on purely morphological .

Introduction of biological diversity (Vodă et al., 2015). Evolutionary lineages which have a recent divergence time but cannot be Accurate species identification is crucial for differentiated morphologically are the result of a process biodiversity studies that aim to characterize patterns of species called cryptic (Bickford et al., 2007). On the other richness and abundance, and to identify recent changes in hand, species may exhibit , a phenomenon these patterns as a result of anthropogenic activities (Austen et whereby conspecific individuals display marked variation in al., 2016; Packer et al., 2018). A critical step for biodiversity morphological characters. If these polymorphic characters studies is the reliable identification of collected individuals are used for species identification, the same evolutionary to the species level. In some cases, independent evolutionary unit may be assigned to different species, leading to an units with very similar morphological characters might be overestimation of biodiversity (Sigovini et al., 2016). When classified as one species leading to an underestimation cryptic or polymorphic species are present in a ,

Open access journal: http://periodicos.uefs.br/ojs/index.php/sociobiology ISSN: 0361-6525 646 C Grando et al. – Color polymorphism in the eucerine bee Melissodes nigroaenea species misidentification may be frequent, especially if dichotomous key the important morphological characters for morphological taxonomic keys are solely used to differentiate species differentiation of South American Melissodes bees, species (Padial et al., 2010). besides had discussed the many taxonomic classifications Even though morphological characters are widely proposed by previous authors. According to the author, there used for species identification, some of the phenotypic traits are two sympatric Melissodes species in Brazil, Melissodes that are easiest to be recognized visually often fail to delimit nigroaenea (Smith) and Melissodes sexcincta (Lepeletier), species accurately (Arribas et al., 2012; Lecocq et al., 2015). whose females can be differentiated mainly by the fullness For example, several studies on insects, including (Grella of the bristle band on the second tergum (TII), but also by et al., 2015), (Garzón-Orduña et al., 2018), and the color of mesosoma hair, and the presence of maxillary bees (Carolan et al., 2012; Ferrari & , 2014; Huang et al., subapical yellow spots. 2015) have shown that traits like color can be hard to interpret In a recent survey of bee in cotton as diagnostic characters for species identification. Color traits ( hirsutum) farmlands from three regions in may exhibit high rates of polymorphism due to environmental Brazil, we collected a large number of female bees that were influences or genetic differences at a single single or multiple identified as M. nigroaenea based in the dichotomous key loci (Miyanaga et al., 1999; Uy et al., 2009; White & Kemp, of Urban (1973). Our specimens showed two distinct color- 2016; Hines et al., 2017). Still, color is commonly used as a morphs of mesosoma (yellow and black) in all populations, trait in taxonomic keys for many , including bees, and few individuals showed variation in the bristle band in , and among many others (Michener, 2007; TII that was not expected for M. nigroaenea. However, the McKay et al., 2014; Rodrílguez et al., 2015). proper identification of the individuals with these unexpected The advent of has led to the patterns of bristle bands was not supported by the use of the development of diagnostic techniques that complement mesosoma color character, what brought us some concern morphological species identification and may ameliorate some about dealing or not with more than one evolutionary unit in of the challenges with morphological taxonomic delimitation. our bee sampling. DNA barcoding is a common molecular technique for species Using the DNA barcoding approach and the phylogenetic and involves the amplification of a highly , in which we consider a species as the variable region of the genome through PCR (polymerase smallest aggregation of sexual population or assexual lineages chain reaction) (Hajibabaei et al., 2007). In , the that presents a unique combination of character states (Wheeler mitochondrial gene cytochrome c oxidase subunit I (COI) is & Platnick, 2000), we tested alternative hypotheses about the marker of choice for DNA barcoding because it can be the interpretation of color variation in M. nigroaenea for easily amplified with universal primers across a large number species identification. Specifically, we investigate whether of taxa (Hebert et al., 2003), and is likely involved in the different color morphs we sampled correspond to one or more process of speciation (Hill, 2016). COI fragments are aligned evolutionary units by quantifying the genetic distance of these and genetic distances between individuals are calculated and individuals using the COI barcoding region. Our hypothesis used as diagnostic boundaries for species. Low genetic distance is that, if significant differences in COI sequences underpin among individuals indicates low divergence suggesting the variation in M. nigroaenea coloration, individuals identified presence of within one evolutionary . as M. nigroaenea actually comprise two evolutionarily On the other hand, large genetic distances among individuals distinct lineages of bees. If instead we observe nominal may indicate the presence of independent evolutionary lineages; between individuals with distinct color- usually identified through a barcoding gap (Hebert et al., morphotypes, our alternative hypothesis is to attribute this 2004; Čandek & Kuntner, 2015). variation to color polymorphism. Despite its known intraspecific variability, color has been a widely used character among bee taxonomists Material and Methods for species descriptions and dichotomous keys (Michener, 2007; et al., 2011; Ascher & Pickering, 2018). Some authors argue that because coloration has a well-characterized M. nigroaenea (Fig 1) is a eucerine bee (Apidae: genetic basis, integument or hair color can be safely used as a Eucerini) distributed along the east coast of Brazil, Uruguay morphological character for species identification (Nemésio, and , and interiorly in Paraguay and Brazil (Fig 2). 2009). However, color polymorphism is well characterized in In Brazil, M. nigroaenea is the most abundant native pollinator several bee groups including bumble bees (Hines & Williams, of cotton (Cardoso et al., 2007), a fiber crop which generated 2012; Lecocq et al., 2015; Duennes et al., 2017), orchid bees $ 74.1 billion USD to the Brazilian economy (Abrapa, 2017). (Ferrari & Melo, 2014) and sweat bees (Miyanaga et al., 1999). This native bee is a highly efficient pollinator of cotton A challenging taxonomic identification involving color , transferring a larger number of grains between characters occurs within the eucerine bees of Melissodes . and generating more per boll when compared A taxonomic description of Urban (1973) organized into a with honey bees (Apis mellifera L.) (Cardoso et al., 2007). Sociobiology 65(4): 645-653 (October, 2018) Special Issue 647 According to Urban (1973), females of M. nigroaenea might mesosoma, showing two complete bristle bands in the TII. be identified for having a predominant black mesosoma with We used these characters to identify 32 female specimens as some variations of yellow, presenting the second bristle brands M. nigroaenea, although we observed few individuals with in the TII incomplete, and the presence of maxillary subapical variation in the bristle bands of TII (almost absent, almost yellow spots. Urban (1973) also describes M. sexcincta as a complete and complete) that did not correspond with the sympatric species, whose females have a predominant yellow description of this species (incomplete)

Fig 1. of the distribution of Melissodes nigroaenea (black dots), and sampling sites of this study (pink squares). source: Urban (1973), Discover (Ascher & Pickering, 2018).

Collection sites do Parecis municipality (Mato Grosso) (Table 1). In 50 x 50 We collected bees in cotton fields between February meter plots within cotton fields, we sampled for bees using two and May 2016 in three different localities in Brazil (Fig 2, methods: a) netting bees visiting cotton flowers while walking pink squares). Collections took place at one site within the along four transects in each plot, and b) setting up four blue Campinas municipality (São Paulo), one site in the Sorriso vane traps filled with propylene glycol. We stored specimens municipality (Mato Grosso), and five sites at Campo Novo in tubes full of 70% ethanol at 10 °C after collection.

Fig 2. Female specimens of Melissodes nigroaenea collected in Mato Grosso, Brazil, representing both mesosoma color morphs (a-b), and showing a continuum in the completeness of TII medial bristle bands (c-f). 648 C Grando et al. – Color polymorphism in the eucerine bee Melissodes nigroaenea Taxonomic identification DNA extraction and quantification Bees were identified by Danielle C. de Luna between We followed a protocol developed by (Boyce et al., May and September 2016 in the Department of Biology at 1989) in to extract genomic DNA from specimens. In Faculty of Philosophy, and Literature of Ribeirão Preto a 1.5 mL tube containing 4 legs of a specimen and 4 metallic at University of São Paulo (FFCLRP/USP). Identifications beads, we added a solution containing 700 mL CTAB 2% were based on the taxonomic key to the genus Melissodes buffer [2% (w/v) CTAB; 1.4 M NaCl, 20 mM EDTA pH 8.00; developed by Urban (1973), and the reference specimens 100 mM Tris-HCl pH 8.00; 1% (w/v) 40,000 PVP; pH 8.3], 2 from the Entomological Collection “Prof. J. M. F. Camargo” µL of -mercaptoethanol and 10 µL of proteinase K at 65 (RPSP), University of São Paulo. °C. Samples were then macerated and incubated at 65 °C for

Table 1. Collection sites, coordinates, color of mesosoma and GenBank accession number of the specimens used in this study. * Individuals with variation in the second bristle band of TII not expected for M. nigroaenea (almost absent, few or complete) Collection Site Coordinate Specimen Color of Mesosoma GenBank Accession Campo Novo do Parecis Fazenda Chapada -13.7873667; -57.5666667 CNCHA_1590 Yellow MH667931 Fazenda Chapada -13.7873667; -57.5666667 CNCHA_BV575 Black MH667928 Fazenda Gaúcha -13.7526781; -57.6417054 CNGAU_1351 Black MH667923 Fazenda Gaúcha -13.7526781; -57.6417054 CNGAU_1362 Black MH667934 Fazenda Gaúcha -13.7526781; -57.6417054 CNGAU_1363 Black MH667924 Fazenda Gaúcha -13.7526781; -57.6417054 CNGAU_1380 Black MH667925 Fazenda Gaúcha -13.7526781; -57.6417054 CNGAU_1387 Yellow MH667926 Fazenda Gaúcha -13.7526781; -57.6417054 CNGAU_1389 Black MH667927 Fazenda Graciosa -13.4755948; -57.9402887 CNGRA_1011 Black MH667906 Fazenda Graciosa -13.4755948; -57.9402887 CNGRA_1013 Black MH667909 Fazenda Graciosa -13.4755948; -57.9402887 CNGRA_1018 Black MH667903 Fazenda Graciosa -13.4755948; -57.9402887 CNGRA_1049 Black MH667912 Fazenda Graciosa -13.4755948; -57.9402887 CNGRA_1061 Yellow MH667904 Fazenda Graciosa -13.4755948; -57.9402887 CNGRA_1066 Yellow MH667907 Fazenda Graciosa -13.4755948; -57.9402887 CNGRA_1072 Yellow MH667910 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1106 Yellow MH667913 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1111 Yellow MH667914 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1112 Black MH667915 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1122* Black MH667916 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1140 Yellow MH667917 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1152 Black MH667918 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1169 Black MH667919 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1184* Black MH667920 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1191* Yellow MH667921 Fazenda Ramada -13.6845448; -57.8804554 CNRAM_1211 Yellow MH667922 Fazenda Santa Teresinha -13.5521948; -57.8435054 CNSTE_925 Black MH667905 Fazenda Santa Teresinha -13.5521948; -57.8435054 CNSTE_994 Black MH667908 Fazenda Santa Teresinha -13.5521948; -57.8435054 CNSTE_997 Yellow MH667911 Sorriso Fazenda Primavera -12.8651948; -55.8820887 SOPP1_BV182* Yellow MH667929 Fazenda Primavera -12.8651948; -55.8820887 SOPP1_BV183 Black MH667932 Campinas Fazenda Santa Elisa -22.8662783; -47.0770554 CPSEL_1626 Yellow MH667933 Fazenda Santa Elisa -22.8662783; -47.0770554 CPSEL_BV588 Black MH667930 Sociobiology 65(4): 645-653 (October, 2018) Special Issue 649 60 min. We then added 600 µL chloroform: isoamyl alcohol Table 2. Taxa, reference paper and GenBank accession number of (24:1) to the tubes and centrifuged at 14,000 rpm for 10 min COI sequences of additional Melissodes species found in GenBank. to separate the aqueous and organic phases, and the upper GenBank aqueous phase transferred to a new 1.5 ml tube. After adding Taxa Reference accession 400 µL of cold isopropanol, the samples were incubated at number Dorchin et al. (2018) MG251042.1 -20 °C for 60 min to precipitate the DNA, followed by a centrifugation at 14,000 rpm for 10 min to collect the DNA Melissodes desponsa Dorchin et al. (2018) MG251044.1 pellet, which was subsequently washed with 70% and 95% Melissodes druriella Hebert et al. (2016) KR801296.1 EtOH. The pellet was then air dried for approximately 2 Melissodes ecuadorius Packer and Ruz (2017) KX820894.1 hours. Finally, 44 µL ultrapure water and 1 µL of pure RNAse Melissodes illata Sheffield et al. (2009) FJ582332 (20 mg/ µL) were added, and the samples incubated for 3 Melissodes paroselae Dorchin et al. (2018) MG251041.1 hours at 37 °C. To approximate the quantify of DNA in the samples, aloquots were run on a 1% (w/v) agarose gel and Results band intensities compared to known amounts in bands of a 100 bp DNA ladder. We identified 13 yellow and 19 black individuals among the 32 samples we collected. Even though Urban (1973) DNA barcoding described the mesosoma color variation as a continuum, we found two discrete coloration (yellow and black, Fig 2a-b) We performed test PCR amplifications with two among the individuals we collected in all populations from combinations of COI primers, Jerry-Pat (Simon et al., 1994) the 3 geographic regions. We also found variation in four and HCO-LCO (Folmer et al., 1994). We made a 25 μL individuals in the completeness of the second bristle band reaction mix containing around 20 ng of genomic DNA, 2 in the TII as described by Urban (1973). Two individuals μL of forward primer (5 pmol/μL), 2 μL of reverse primer presented almost no bristal bands (CNRAM_1122, Fig 2c, and (5 pmol/μL), 0.5 μL of dNTP mix (10 mM), 2.5 μL of 10× CNRAM_1184), one presented few bristle bands in the lateral Standard Taq Reaction Buffer, 2 μL of MgCl (25 mM), 2 (SOPP1_BV182. Fig 2d), and one individual presented complete and 1 U of Taq DNA polymerase (Fermentas, Thermo bristle bands (CNRAM_1191, Fig 2f). Most individuals Scientific, Pittsburgh, Pennsylvania, USA). PCR programs for presented incomplete bristle bands, like CNGAU_1387 (Fig 2e). both primer pairs consisted of an initial denaturation step of PCR reactions with the primer pair LCO 1490/HCO three minutes at 94 ºC, followed by 40 cycles of 30 seconds 2198 were optimized for our specimens at an annealing at 94 ºC, 30 seconds for annealing at the specific temperature temperature of 50° C, producing a single distinct band at for each primer (tested with 48, 50, 51 and 52 ºC) and 1 710 bp. The primer pair Jerry-Pat failed to amplify from min at 72 ºC, followed by a final extension step of 10 min our specimens. We identified GTR+G as the best nucleotide at 72 ºC. We used a 1% (w/v) agarose gel to corroborate the substitution model for our data. The consensus obtained amplification products. We used a Qiagen PCR purification from MrBayes shows that the COI fragments group both color kit to clean the PCR products and used bidirectional Sanger morphs into one highly supported (Posterior Probability sequencing at the University of Illinois at Urbana-Champaign = 1) sister to Melissodes ecuadorius (Bertoni and Schrottky), Core Sequencing Facility (USA). a species distributed in Ecuador, Peru, and Chile. Alignment of sequencing data We found that the average pairwise genetic p-distance within yellow individuals was 0.56 % (± 0.20 %), between Raw sequences were edited and aligned in the software black individuals was 0.86 % (± 0.26 %) while between yellow Geneious (Kearse et al., 2012). In addition to the 32 COI and black individuals was 0.73% (± 0.23%) (Table 3). This sequences from our study, we downloaded six COI sequences level of genetic differentiation in mitochondrial DNA does of other Melissodes species from GenBank and added them not support the presence of two evolutionarily independent to the alignment (Table 2). To select the optimal model of units between the different mesosoma colors. The average nucleotide for phylogenetic reconstruction, we used pairwise genetic distances among M. nigroaenea and other the program PartitionFinder2 (Lanfear et al., 2017). We species were between 8% and 11.5% (Table 3). ran MrBayes (Ronquist & Huelsenbeck, 2003) using the best nucleotide model to estimate the phylogenetic relationships Discussion among individuals using a . We adopted a sample size of 107 generations and a burn-in of 25% of the , Both pairwise genetic distances and the Bayesian sampling every 1,000 generations with Markov Chain Monte phylogenetic reconstruction supported that M. nigroaenea Carlo (MCMC) methods, which generated a consensus tree individuals with yellow and black coloration in the mesosoma with high branch support. Our alignment file was also read into belong to the same evolutionary unit (Fig 3a-b; Table 3). MEGA7 (Kumar et al., 2016) to estimate the pairwise genetic Pairwise genetic distances within and between the two p-distance between our samples using 10,000 bootstraps. color-morphs were below 3%, considered a threshold for 650 C Grando et al. – Color polymorphism in the eucerine bee Melissodes nigroaenea differentiating species in other orders (Hebert et al., of variation in the mesosoma coloration in M. nigroaenea, 2003). These results indicate that the yellow and black color suggesting possible intergradation in this diagnostic character. morphs are a polymorphism present in sympatric populations Another taxonomic character mentioned by Urban (1973) to of M. nigroaenea. Additionally, we also observed significant differentiate females of both species was the bristle bands variation in the bristle band fullness of TII in four individuals, in the TII, in which we observed a continuous variation not but the variation in this character was more continue than the expected for M. nigroaenea, from the almost absence to the observed variation in the color of the mesosoma (Fig 3c-f). fullness of the second bristle band in TII. We identified four Taxonomic delimitations in the Eucerini can be variants for this character, of which three are from Fazenda challenging primarily due to the morphological intergradation Ramada (Campo Novo do Parecis region), and one from — meaning presence of intermediate characters (Terrell, Fazenda Primavera (Sorriso region), that do not constitute a 1963) — among taxa (Michener, 2007; Dorchin et al., 2018). separated evolutionary unit based in our analysis. Although In the Melissodes species description proposed by Urban we might suggest the morphological intergradation for this (1973), the color of the mesosoma is an important character to character, further studies with more individuals from different differentiate female M. nigroaenea (black) from M. sexcincta regions will support if the bristle bands in TII could also be (yellow). However, this same author recognizes the presence considered a polymorphic character for M. nigroaenea.

Fig 3. Consensus based on Bayesian inference generated from COI sequences for the 32 yellow and black morphs studied, plus six additional COI sequences for other Melissodes genus found in GenBank. The numbers over the branches correspond to posterior probability values. The scale in the bottom (0.03) means the number of substitutions per site. Sociobiology 65(4): 645-653 (October, 2018) Special Issue 651 Table 3. Average pairwise genetic p-distance between each color morph and other Melissodes species, with their respective average standard error estimates. (*) Average pairwise genetic p-distance within each color morph, with their respective average standard error estimates. Melissodes Melissodes Melissodes Melissodes Melissodes Melissodes Melissodes Melissodes nigroaenea nigroaenea ecuadorius illata desponsa druriella paroselae agilis black yellow Melissodes 0.86 % 0.73 % 8.03 % 9.74 % 11.00 % 11.00 % 11.30 % 11.50 % nigroaenea (± 0.26 %)* (± 0.23 %) (± 1.18 %) (± 1.31 %) (± 1.34 %) (± 1.39 %) (± 1.43 %) (± 1.42 %) black Melissodes 0.73 % 0.56 % 8.01 % 9.59 % 10.87 % 10.90 % 11.29 % 11.50 % nigroaenea (± 0.23 %) (± 0.20 %)* (± 1.19 %) (± 1.31 %) (± 1.35 %) (± 1.39 %) (± 1.44 %) (± 1.43 %) yellow

Urban (1973) also made a description about the in the TII might be considered a polymorphic character. dentiform male genitalia for Melissodes species identification. In addition, more molecular markers will be necessary to According to the author, males of M. nigroaenea presents the determine whether these two color-morphs are the result of genitalia with a cut near the valve, while M. sexcincta does recent incipient speciation. Species delimitation studies using not present this cut. However, despite of the reliance on male museum materials identified as M. sexcincta may assist in genital characters to confirm the taxonomy in South American clarifying whether this species and M. nigroaenea and are two Melissodes bees, our samples were composed mostly by evolutionary independent entities or should be synonymized. females, while the few males we found were not presented in all populations. Thus, this morphological character was Acknowledgements not considered for analyses, but may merit further study, especially in combination with DNA barcoding. We would like to thank to São Paulo Research Additional studies using molecular techniques to Foundation - FAPESP for funding this research (Grant number resolve taxonomic issues within bees have also concluded 2014/50738-9), and the Coordination for the Improvement of that color should not be used as a primary character for Higher Education Personnel - CAPES (Programa Biologia taxonomic classification. Ferrari and Melo (2014) found that Computacional, Brazil grant number 1572813, and US Grant the integument color of three species of orchid number 060/2017-08) for the Post-doctoral scholarship of C. bees present geographic variation in Brazil, with both blue Grando at University of Campinas and Penn State University, and green colors observed. However, the authors did not respectively. We thank to Danielle C. de Luna for the find phylogenetic support for the independence of the three precious help with bee collections and the identifications, as Euglossa species, nor did they observe a vast enough genetic well as Prof. Eduardo A. B. de Almeida for the use of his distance to justify separating both color morphs into different lab and the access to the Entomological Collection “Prof. J. species. Another group of bees known for high variability in M. F. Camargo” (RPSP), University of São Paulo, during the coloration pattern are the bumble bees. Huang et al. (2015) identification. We are also grateful to Instituto Matogrossense found nine distinct color patterns for female bumble bees do Algodão (IMAmt) for making possible our bee collections in different regions of , but all color morphs clustered in cotton farms of the State of Mato Grosso (Brazil). Last, but together as a monophyletic group in the Bayesian analysis, extremely important, we would like to express our admiration belonging to Bombus koreanus (Skorikov). Carolan et al. and gratitude for all the contributions of Prof. Danuncia Urban (2012) tested the hypothesis that color patterns could reliably for the taxonomy of bees in Brazil. distinguish three Bombus in the lucorum complex from one another when compared to COI sequence data, and found that Authors’ Contributions morphological data often resulted in incorrect classification of CG, MMLU and MIZ conceived and designed the individual specimens, due to the cryptic of the lucorum study; CG conducted the field sampling; CG, SC, NG and complex. However, taxonomic characters can be informative WW generated the molecular data; PA photographed bee to identify species, especially when they are interpreted in individuals; CG and NDA conducted the data analyses; CG, conjunction with another of dataset like DNA barcoding NDA, MIZ, and MMLU drafted the manuscript. All authors (Freitas et al., 2018). gave final approval of the manuscript for publication. Here, we confirmed a mesosoma color polymorphism in M. nigroaenea, an important pollinator of cotton plantations References in Brazil. Our results suggest that this species exhibits yellow and black individuals in multiple areas across its distribution. Arribas, P., Abellán, P., Velasco, J., Bilton, D.T., Millán, Sampling a higher number of individuals from more populations A. & Sánchez-Fernández, D. (2012). 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